ABSTRACT Genital herpes simplex virus-2 (HSV-2) infections are a major public health problem affecting >40 million people in the United States and >500 million globally. HSV-2 infection is characterized by painful recurring, genital sores and this can coincide with considerable psychological distress. Transmission can occur during sexual contact and from mother to child during birth with fatal consequences. Although several vaccines have been shown to elicit strong neutralizing activity against the virus, there is currently no effective vaccine to prevent HSV-2. Recent research has shown that while systemic, humoral immunity is beneficial, co-induction of mucosal immunity is prerequisite for full protection. BlueWillow Biologics has established NanoVax, a proprietary oil-in- water nanoemulsion (NE01) adjuvant technology, that is designed for intranasal immunization. We've demonstrated the ability of NE01 to augment the targeting of antigens to the immune system while safely eliciting potent humoral, mucosal and cellular immune responses, including balanced induction of Th1, Th2 and Th17 cells. This feature facilitates the development of efficacious vaccines that induce mucosal and cell mediated immunity for full protection against various respiratory and sexually transmitted pathogens, including HSV-2. To address this unmet medical need, BlueWillow Biologics is developing a novel, intranasal bivalent vaccine comprised of two HSV-2 glycoproteins (gB2 and gD2) formulated in NE01. Our preclinical studies have shown that the resulting intranasal vaccine (IN) gB2/gD2-NE01 is an effective and commercially viable prophylactic strategy to prevent HSV-2 infections. We have optimized the processes for scale-up manufacturing of the antigens to achieve reproducible production of highly pure gB2 and gD2 drug substances at a scale sufficient to meet clinical demand. Importantly, we've demonstrated the prophylactic equivalency of the drug substances prepared from the established scale-up manufacturing processes to those we previously prepared at lab scale. This demonstrates our ability to manufacture high quantity of pure material while preserving the integrity and activities of the key vaccine components. In this Phase IIB SBIR program, BlueWillow Biologics will execute the remaining development work on the IN gB2/gD2-NE01 HSV-2 vaccine necessary to submit an investigational new drug (IND) application. Using the established scale-up processes established under Phase II, we will refine the gB2 expression process to improve yield, manufacture two batches of each antigen, along with the NE01 adjuvant, and dedicate the material towards completing an IND-enabling GLP toxicology study and formulating clinical material for stability assessment. The preparation of this material will enable a pre-IND meeting with the FDA to request feedback and approval of our GLP toxicology study design. Upon completion of the GLP toxicology study, BlueWillow will be ready to file an IND application and initiate a Phase 1 clinical trial (not covered under this funding).